A 50 kDa Protein Modulates Guanine Nucleotide Binding of Transglutaminase II

Abstract

Regulation of cellular response is an important mechanism for controlling cellular functions. The transmembrane signaling of the hormone receptors is regulated by GTP-binding proteins (GTPases) and their associated proteins. Our previous studies demonstrated that the bifunctional GTP-binding protein, Gαh (transglutaminase II), consistently copurified with an ∼50 kDa protein (Gβh) which is dissociated from Gαh upon activation with GTPγS or AlF4-. Present immunological and biochemical studies on the regulation of the GTPase cycle of Gαh, which involves the α1-adrenoceptor and 50 kDa Gβh, reveal that the 50 kDa protein is indeed a Gαh-associated protein and down-regulates functions of Gαh. Thus, polyclonal antibody against Gβh coimmunoprecipitates GDP-bound Gαh but not the GDP−AlF4--bound form. The GTPγS binding and GTPase activity of Gαh are inhibited in a Gβh concentration dependent manner. Supporting this notion, Gβh accelerates GTPγS release from Gαh and changes the affinity of Gαh from GTP to GDP. Moreover, the ternary complex preparation exhibits TGase activity that is inhibited in the presence of the α1-agonist and GTP. The GTPγS binding by the ternary complex, consisting of the α1-agonist, the receptor, and Gh, is also inhibited by Gβh. The inhibition of GTPγS binding with the ternary complex requires a ≥2.7-fold higher concentration of Gβh than that for Gαh alone, indicating that the receptor enhances the affinity of Gαh for GTP. In addition, Gβh copurifies with an α1-agonist, adrenoceptor, and Gαh ternary complex, showing that the complex is a heterotetramer. Our data also suggest that Gβh does not directly interact with the α1-adrenoceptor. These findings clearly demonstrate that Gαh associates with a novel protein which modulates the affinity of Gαh for guanine nucleotides and that the GDP-bound Gh is the ground state for the counterpart activator, the α1-adrenoceptor, in this signaling system.